There is an interest in the state of the art to obtain transport systems which, maintaining the integrity and viability of the cells, do not requiring handling complicated methodologies, both during their transport and during the process for the recovery of the cells, in their final destination.
Cell transport is currently carried out in two different ways, which is by transporting cells in a cryopreserved state or in a cultured state.
The techniques for the transport of cells in culture allow the cells to be adhered or in suspension in flasks with liquid culture medium. In this methodology, it is necessary to be extremely careful with the transport conditions because small movements sustained over time during their transport definitively affect the integrity and cell adhesion capacity and therefore the viability of the cells in their destination. This means that most of the cultures thus transported do not reach their place destination in conditions of viability that are suitable for being used in the various research projects, understanding cell viability as there being no morphological and/or functional alterations in the cells.
This is why an effective cell culture transport system must assure that during the entire transport process and the process for the recovery of the cells for their use, the integrity thereof is optimally maintained (references 15-19), i.e., the viability thereof is not affected during the entire process.
Transport in the cryopreserved state involves the transport of vials of cells in a cryofrozen state, which means that in order for the cells to be used again, the place of destination must have installations and personnel that are specialized in cell culture, a tedious manipulation including cell amplification and maintenance, and the arrangement of the cells in the formats required for carrying out the testing techniques, being necessary.
So one aspect to take into account in cell transport is the temperature at which the cells are transported, because this temperature directly affects the maintenance of the cell integrity due to the fact that most cell lines and types are sensitive to temperature changes.
Cryopreservation involves very low and constant transport temperatures that are difficult to maintain the entire transport time. This means that transport must take place in very specific conditions, i.e., the vials must be kept at temperatures of less than −80° C. during the entire transport process because cell viability would otherwise be seriously affected.
The optimal growth temperature for animal cell cultures is 36-38° C. Once this temperature range is exceeded (hyperthermia conditions) cell viability is affected, irreversibly damaging the integrity of the cells of the culture and causing cell death.
Temperatures under the optimal temperature range (hypothermia conditions) are better tolerated by cell cultures than high temperatures are. So, in the case of the application of a temperature of less than the optimal recommended temperature, decreased cell metabolism occurs, i.e., the cell reactions (proliferation, metabolisms, growth, . . . ) slow down but the cell maintains its integrity, and when optimal thermal conditions for growth are restored, the cells recover their cell activity.
In the state of the art, hypothermia is a widely used methodology for slowing down the growth of microorganisms and tumor cells. The system described in the present invention makes use of said characteristic such that the cells have slowed metabolism during transport, aiding in the maintenance of their integrity.
The state of the art comprises several systems for the transport of cells in culture. In certain documents, the cell cultures are covered with culture media at 1-20% of liquid gelatin which, after solidification, can be transported without the cells being damaged. Other documents, however, describe specific devices for the culture, storage and transport of cell cultures.
Patent application P200301526 describes a method for the storage and transport of two-dimensional cell cultures in which the cells are immobilized on a transwell-type asymmetric support which is covered with a gelatin solution at a concentration of 1 to 5% which solidifies by cooling, thus facilitating that the system can be transported, maintaining the cell integrity of the culture. The plate is incubated in the laboratory of destination at 37° C. for 4 hours so that the gelatin liquefies and can be removed from the cell culture, the cells being ready to perform the appropriate migration assays. This system, however, does not allow using the cell culture for applications other than assays in transwell-type asymmetric supports.
In contrast, the invention proposed in the present document is suitable for any cell culture format required according to its specific application either in the transport system itself or once it is extracted therefrom and allows cell recovery to occur in a time of not more than 3 hours, which is less time than that described by Spanish patent application P200301526 (4 hours).
European patent EP0702081 describes a method for the storage and transport of three-dimensional tissues. The invention described in this document consists of placing a three-dimensional culture of skin fixed on two types of sponge covered with a gelatin solution of 1-20%, preferably of 5-10%, such that when the solution gels by cooling, this facilitates its transport and storage. As in the aforementioned Spanish patent application, this document describes the method used to remove the gelatin from the three-dimensional culture which consists of increasing the temperature up to a maximum of 37° C. to liquefy it, preserving the cell integrity of the system. The document specifies that the use of agarose would not be suitable for this system because the melting point of agarose is around 60° C., i.e., much higher than the temperature allowing cell viability. The system described by this document furthermore does not allow the recovery of the cell culture for its use outside same.
International patent application W02007/080600 describes a disposable device for the culture and/or storage and transport of viable adherent cells. In said device, the cells are seeded on membranes, gels or microporous substrates held to the device on which a medium providing the cells with the nutrients necessary for maintaining their growth will later be added. The device closes securely, preventing losses of liquid and the entrance of air in the compartment in which the cells are cultured. Once the device reaches its destination, the membrane, gel or substrate on which the cells are seeded is extracted and cleaned and it can then be transplanted. However, the document does not indicate in any case that the cells are separate from the membrane on which they are cultured, such that such cells cannot be used independently. The device described by this document furthermore does not allow the transport of cell cultures which are not adherent.
Therefore, the present invention presents as a transport medium an agarose and agarase solution which gels at room temperature and which can be removed once the sample reaches its destination after heating the system to 37° C., as a result of the activation of the agarase at said temperature which facilitates the digestion of agarose. The system furthermore is suitable for any culture format, including plates, culture chambers, bottles, tubes, transwell-type asymmetric supports, etc.
The transport system mentioned in the present invention assures that the cells can be transported in culture, both adhered and in suspension, preventing that the movements derived from transport damage cell integrity and therefore maintaining optimal cell viability that allows recovering the transported cells or carrying out different types of assays with them.
Agarose is a thermally reversible polysaccharide consisting of alternating (1-3) linked β-D-galactose and (1-4) linked (3-6)-anhydrous-α-L-galactose copolymers, and it is commonly used in cell encapsulation. Agarose can be melted or gelled through changes in the temperature to which it is subjected.
Cell encapsulation in agarose is known in the state of the art for carrying out a wide variety of applications, such as for example the use of cells as biosensors, for therapeutic uses, etc. (references 11, 12). In addition, the biocompatibility the agarose has been proven by means of in vivo implantation studies (references 1, 2, 13). It has furthermore been observed that the cells that have been encapsulated in agarose hydrogels have the capacity to secrete their own extracellular matrix (reference 14) which reflects that the functional behavior of the cells is not altered in this medium. However, agarose is not a medium commonly used in forming three-dimensional cultures, since it does not seem to induce cell proliferation in this type of cell culture.
Agarase is an enzyme with a molecular weight of 32 kDA that hydrolyzes the [1-3] linkages between D-galactose and 3,6-anhydrous-L-galactose residues of agarose.
The state of the art also describes how cultures encapsulated with agarose can be recovered by means of treatment with an agarase solution added on the culture. In these cases the receiving laboratory must have agarase, prepare the mixture at the necessary concentration and adding it to the system. However the digestion of agarose following this method is not homogenous because the agarase is not in direct contact with all the agarose gel, which increases the recovery time, finally reducing cell viability.
An example can be found in references 1 and 2, which show the recovery of cell cultures embedded in a 1.5% agarose solution. To carry out said recovery, the three-dimensional structure is treated with an agarase solution that is added independently. Document WO2001/40445 uses a 2% agarose solution to treat a cell or cell populations and to capture the substances said cells secrete. The agarose used is previously treated to incorporate cytokine- and hormone-specific binding sites therein. To remove the agarose matrix, an agarase solution causing the enzymatic digestion of agarose is added.
In contrast, the system of the present invention involves the use of a homogenous agarose-agarase mixture as the transport medium, which prevents the receiving laboratory from having to have agarase, prepare the mixture at the necessary concentration and add it to the system. Furthermore, the system of the present invention favors a homogenous digestion throughout the entire cell culture, achieving optimal viability of the recovered cell cultures.
An additional advantage of the system of the present invention is the fact that it uses lower percentages of agarose, which means that during cell recovery, the amount of agarase necessary for the complete digestion of agarose is lower and therefore the cells will not be affected by its enzymatic action, showing no alterations in terms of the viability and proliferative capacity thereof.
In view of the state of the art, there is obviously a need to provide a standard cell transport system for both adherent and non-adherent cell cultures, suitable for any cell culture format and application of the system, which assures the integrity and viability of the cell culture during the transport process, and that the cell recovery process does not require installations or personnel specialized in cell culture, is obvious. The transport system described in the present invention proposes a simple cell transport system which allows transporting cells in culture, both adhered and in suspension, with maximum quality standards and viability, and furthermore does not necessarily require a specific infrastructure for recovering and using the cell culture if the final application of the transported cells does not require this.
The transport conditions required by the system described by the present invention are not conditions that require a cooled transport temperature, and the transport time is not a risk factor either because the cell transport system of the invention allows the cells to be transported at temperatures of not more than 25° C. during a broad time interval without affecting the viability of the cell culture.